Multidimensional cluster stability analysis from a Brazilian Bradyrhizobium sp RFLP/PCR data set

The taxonomy of the N(2)-fixing bacteria belonging to the genus Bradyrhizobium is still poorly refined, mainly due to conflicting results obtained by the analysis of the phenotypic and genotypic properties. This paper presents an application of a method aiming at the identification of possible new clusters within a Brazilian collection of 119 Bradryrhizobium strains showing phenotypic characteristics of B. japonicum and B. elkanii. The stability was studied as a function of the number of restriction enzymes used in the RFLP-PCR analysis of three ribosomal regions with three restriction enzymes per region. The method proposed here uses Clustering algorithms with distances calculated by average-linkage clustering. Introducing perturbations using sub-sampling techniques makes the stability analysis. The method showed efficacy in the grouping of the species B. japonicum and B. elkanii. Furthermore, two new clusters were clearly defined, indicating possible new species, and sub-clusters within each detected cluster. (C) 2008 Elsevier B.V. All rights reserved.

Expression analysis of putative vitellogenin and lipophorin receptors in honey bee (Apis mellifera L.) queens and workers

Two members of the low density lipoprotein receptor (LDLR) family were identified as putative orthologs for a vitellogenin receptor (Amvgr) and a lipophorin receptor (Amlpr) in the Apis mellifera genome. Both receptor sequences have the structural motifs characteristic of LDLR family members and show a high degree of similarity with sequences of other insects. RT-PCR analysis of Amvgr and Amlpr expression detected the presence of both transcripts in different tissues of adult female (ovary, fat body, midgut, head and specifically hypopharyngeal gland), as well as in embryos. In the head RNA samples we found two variant forms of AmLpR: a full length one and a shorter one lacking 29 amino acids in the O-linked sugar domain. In ovaries the expression levels of the two honey bee LDLR members showed opposing trends: whereas Amvgr expression was upregulated as the ovaries became activated, Amlpr transcript levels gradually declined. In situ hybridization analysis performed on ovaries detected Amvgr mRNA exclusively in germ line cells and corroborated the qPCR results showing an increase in Amvgr gene expression concomitant with follicle growth. (C) 2008 Elsevier Ltd. All rights reserved.

Prevalence of ehrlichial infection among dogs and ticks in Northeastern Brazil

This study investigated the epidemiology of canine ehrlichiosis in Northeastern Brazil, focusing the identification of the Ehrlichia species and vectors involved. Samples were collected from 472 domestic dogs residing in the health districts of Cajazeiras and Itapuã of Salvador city. The average prevalence of antibodies reactive to E. canis by immunofluorescent antibody test (IFAT) (titer > 1:80) was 35.6% (168/472). Blood samples from the E. canis-seropositive animals were tested by nested PCR in order to identify the Ehrlichia species responsible for the infection. Among the seropositives, 58 (34.5%) were found to be PCR-positive for E. canis. Ticks were found in 32 dogs. Nested-PCR analysis showed that 21.9% (7/32) of the Rhipicephalus sanguineus were infected by E. canis. In both dogs and Rhipicephalus sanguineus, nested-PCR for E. ewingii and E. chaffeensis was negative, with no amplification of DNA fragment.

Hormone-regulated expression and distribution of versican in mouse uterine tissues

Background: Remodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination. Methods: Uteri from mice in all phases of the estrous cycle, and animals subjected to ovariectomy and hormone replacement were collected for immunoperoxidase staining for versican, as well as PCR and quantitative Real Time PCR. Results: In diestrus and proestrus, VER was exclusively expressed in the endometrial stroma. In estrus and metaestrus, VER was present in both endometrial stroma and myometrium. In OVX mice, VER immunoreaction was abolished in all uterine tissues. VER expression was restored by E2, MPA and E2+MPA treatments. Real Time PCR analysis showed that VER expression increases considerably in the MPA-treated group. Analysis of mRNA identified isoforms V0, V1 and V3 in the mouse uterus. Conclusion: These results show that the expression of versican in uterine tissues is modulated by ovarian steroid hormones, in a tissue-specific manner. VER is induced in the myometrium exclusively by E2, whereas MPA induces VER deposition only in the endometrial stroma.

LOW-INTENSITY PULSED ULTRASOUND PRODUCED AN INCREASE OF OSTEOGENIC GENES EXPRESSION DURING THE PROCESS OF BONE HEALING IN RATS

The aim of this study was to measure the temporal expression of osteogenic genes during the process of bone healing in low-intensity pulsed ultrasound (LIPUS) treated bone defects by means of histopathologic and real-time polymerase chain reaction (PCR) analysis. Animals were randomly distributed into two groups (n = 30): control group (bone defect without treatment) and LIPUS treated (bone defect treated with LIPUS). On days 7, 13 and 25 postinjury, 10 rats per group were sacrificed. Rats were treated with a 30 mW/cm(2) LIPUS. The results pointed out intense new bone formation surrounded by highly vascularized connective tissue presenting a slight osteogenic activity, with primary bone deposition was observed in the group exposed to LIPUS in the intermediary (13 days) and late stages of repair (25 days) in the treated animals. In addition, quantitative real-time polymerase chain reaction (RT-qPCR) showed an upregulation of bone morphogenetic protein 4 (BMP4), osteocalcin and Runx2 genes 7 days after the surgery. In the intermediary period, there was no increase in the expression. The expression of alkaline phosphatase, BMP4 and Runx2 was significantly increased at the last period. Our results indicate that LIPUS therapy improves bone repair in rats and upregulated osteogenic genes, mainly at the late stages of recovery. (E-mail: a.renno@unifesp.br) (C) 2010 Published by Elsevier Inc. on behalf of World Federation for Ultrasound in Medicine & Biology.

Histological, physiological and molecular investigations of Fagus sylvatica seedlings infected with Phytophthora citricola

P>The aim of the work was to shed light into histological, physiological and molecular changes of Fagus sylvatica seedlings infected with the root pathogen Phytophthora citricola with the final goal to distinguish between local and systemic responses. Real-time quantitative PCR analysis proved that P. citricola was able to grow from infected roots into hypocotyl and epicotyl tissue of F. sylvatica seedlings. Light microscopy showed many collapsed parenchyma cells of the cortex without being penetrated by the pathogen. Hyphae were mainly growing intracellular in parenchyma and xylem tissue. Transmission electron microscopy displayed disintegration of xylem vessels and of parenchyma cells. Inhibition of water uptake of infected beech seedlings was positively correlated with the concentration of zoospores used in the experiment. In addition, a split root experiment indicated that invertases were possibly involved locally and systemically in the conversion of sucrose of P. citricola infected roots. During the growth of the pathogen in roots, a transient expression of the 1-aminocyclopropane-1-carboxylic acid (ACC)-oxidase gene was quantified in leaves which was detected in parallel with the first peak of a biphasic ethylene outburst. Additionally a systemic upregulation of aquaporin transcripts was mainly detected in leaves of beech seedlings infected with P. citricola.

Short communication: An evaluation of the effectiveness of Lactobacillus buchneri 40788 to alter fermentation and improve the aerobic stability of corn silage in farm silos

The objective of this study was to determine if the effects of inoculation with Lactobacillus buchneri 40788 were detectable when applied to whole-plant corn stored in farm silos. Corn silage was randomly sampled from farms in Wisconsin, Minnesota, and Pennsylvania, and was untreated (n = 15) or treated with an inoculant (n = 16) containing L. buchneri 40788 alone or this organism combined with Pediococcus pentosaceus during May and June 2007. Corn silage that was removed from the silo face during the morning feeding was sampled, vacuum-packed, and heat sealed in polyethylene bags and shipped immediately to the University of Delaware for analyses. Silage samples were analyzed for dry matter (DM), nutrient composition, fermentation end-products, aerobic stability, and microbial populations. The population of L. buchneri in silages was determined using a real-time quantitative PCR method. Aerobic stability was measured as the time after exposure to air that it took for a 2 degrees C increase above an ambient temperature. The DM and concentrations of lactic and acetic acids were 35.6 and 34.5, 4.17 and 4.85, and 2.24 and 2.41%, respectively, for untreated and inoculated silages and were not different between treatments. The concentration of 1,2-propanediol was greater in inoculated silages (1.26 vs. 0.29%). Numbers of lactic acid bacteria determined on selective agar were not different between treatments. However, the numbers of L. buchneri based on measurements using real-time quantitative PCR analysis were greater and averaged 6.46 log cfu-equivalents/g compared with 4.89 log cfu-equivalent for inoculated silages. There were fewer yeasts and aerobic stability was greater in inoculated silages (4.75 log cfu/g and 74 h of stability) than in untreated silages (5.55 log cfu/g and 46 h of stability). This study supports the effectiveness of L. buchneri 40788 on dairy farms.

The insulin signaling pathway in honey bee (Apis mellifera) caste development - differential expression of insulin-like peptides and insulin receptors in queen and worker larvae

The insulin/insulin-like signaling (IIS) pathway is an evolutionarily conserved module in the control of body size and correlated organ growth in metazoans. In the highly eusocial bees, the caste phenotypes differ not only in size and several structural features but also in individual fitness and life history. We investigated the developmental expression profiles of genes encoding the two insulin-like peptides (AmILP-1 and AmILP-2) and the two insulin receptors (AmInR-1 and AmInR-2) predicted in the honey bee genome. Quantitative PCR analysis for queen and worker larvae in critical stages of caste development showed that AmILP-2 is the predominantly transcribed ILP in both castes, with higher expression in workers than in queens. Expression of both InR genes sharply declined in fourth instar queen larvae, but showed little modulation in workers. On first sight, these findings are non-intuitive, considering the higher growth rates of queens, but they can be interpreted as possibly antagonistic crosstalk between the IIS module and juvenile hormone. Analyzing AmInR-1 and AmInR-2 expression in ovaries of queen and worker larvae revealed low transcript levels in queens and a sharp drop in AmInR-2 expression in fifth instar worker larvae, indicating relative independence in tissue-specific versus overall IIS pathway activity. (C) 2008 Elsevier Ltd. All rights reserved.

Glycodelin expression in the endometrium of healthy women and in the eutopic and ectopic endometrium of women with endometriosis

Objective: To analyze the expression of the glycodelin gene to better understand the molecular environment of endometriotic lesions and to elucidate the potential mechanisms that underlie the complex physiopathology of endometriosis. Design: Prospective laboratory study. Setting: University hospital. Patient(s): Eleven healthy fertile women and 17 patients with endometriosis in the early proliferative phase of the menstrual cycle. Intervention(s): Endometrial biopsy specimens were obtained from the endometrium of healthy women without endometriosis (controls) and from eutopic and ectopic endometrium tissues (pelvic and ovarian endometriotic implants) of endometriosis patients. Main Outcome Measure(s): The glycodelin relative expression level by real-time polymerase chain reaction (PCR) analysis. Result(s): The glycodelin down-regulation found in the endometriotic lesions was 332.26 and 123.17-fold lower, respectively, when compared with the eutopic tissue and the control endometrium. Conclusion(s): Glycodelin may be one of the molecules that contributes to the loss of cellular homeostasis in endometriotic lesions. (Fertil Steril (R) 2009;91:1676-80. (C)2009 by American Society for Reproductive Medicine.)

Cryptic SYT/SXX1 fusion gene in high-grade biphasic synovial sarcoma with unique complex rearrangement and extensive BCL2 overexpression

Synovial sarcomas are high-grade malignant mesenchymal tumors that account for 10% of all soft-tissue sarcomas. Almost 95% of these tumors are characterized by a nonrandom chromosomal abnormality, t(X;18)(p11.2;q11.2), that is observed in both biphasic and monophasic variants. In this article, we present the case of a 57-year-old woman diagnosed with high-grade biphasic synovial sarcoma in which conventional cytogenetic analysis revealed the constant presence of a unique t(18;22)(q12;q13), in addition to trisomy 8. The rearrangement was confirmed by fluorescence in situ hybridization. The use of the whole chromosome painting probes WCPX did not detect any rearrangements involving chromosome X, although reverse-transcriptase polymerase chain reaction (PCR) analysis demonstrated the conspicuous presence of a SYT/SXX1 fusion gene. Spectral karyotyping (SKY) was also performed and revealed an insertion of material from chromosome 18 into one of the X chromosomes at position Xp11.2. Thus, the karyotype was subsequently interpreted as 47,X,der(X)ins(X;18) (p11.2;q11.2q11.2),der(18)del(18)(q11.2q11.2)t(18;22)(q12;q13),der(22)t(18;22). Real-time PCR analysis of BCL2 expression in the tumor sample showed a 433-fold increase. This rare finding exemplifies that thorough molecular-cytogenetic analyses are required to elucidate complex and/or cryptic tumor-specific translocations. (C) 2010 Elsevier Inc. All rights reserved.

Tick toxicosis in a dog bitten by Ornithodoros brasiliensis

Ticks are hematophagous parasites of people and animals and are a public health hazard in several countries. They are vectors of infectious diseases; in addition, the bite of some ticks, mainly from the Ornithodoros genus, may lead to local lesions and systemic illness, referred to as tick toxicosis. In this report, we describe a dog bitten by Ornithodoros brasiliensis, popularly known as the mouro tick. The main clinical findings were disseminated skin rash, pruritus, mucosal hyperemia, lethargy, and fever. Laboratory abnormalities 48 hours after the bites occurred included mild nonregenerative anemia, eosinophilia, basophilia, increased serum creatine kinase activity, increased serum C-reactive protein concentration, and prolonged coagulation times. Tick-borne pathogens were not detected by PCR analysis or serologic testing, supporting the diagnosis of a noninfectious syndrome due to tick bite, compatible with tick toxicosis.

Expression of Extracellular Matrix Proteins in Human Dental Pulp Stem Cells Depends on the Donor Tooth Conditions

Introduction: Stem cells are characterized by the ability to renew themselves through mitotic cell division and differentiating into a diverse range of specialized cell types. An important source of adult stem cells is the dental pulp. In dentistry, regenerative strategies are of importance because of hard dental tissue damage especially as result of caries lesions, trauma, or iatrogenic procedures. The regeneration of dental tissues relies on the ability of stem cells to produce extracellular (ECM) proteins encountered in the dental pulp tissue. Thus, the aim of this study was to analyze the expression and distribution of proteins encountered in dental pulp ECM (type I collagen, fibronectin, and tenascin) in stem cells. Methods: Human immature dental pulp stem cells (hIDPSCs) from deciduous (DL-1 and DL-4 cell lines) and permanent (DL-2) teeth were used. The distribution of ECM proteins was observed using the immunofluorescence technique. The gene expression profile was evaluated using reverse transcription polymerase chain reaction (RT-PCR) analysis. Results: Positive reactions for all ECM proteins were observed independently of the hIDPSCs analyzed. Type I collagen appeared less evident in DL-2 than in other hIDPSCs. Fibronectin and tenascin were less clear in DL-4. The RT-PCR reactions showed that type I collagen was lesser expressed in the DL-2 cells, whereas fibronectin and tenascin were similarly expressed in all hIDPSCs. Conclusions: The distribution and expression of ECM proteins differ among the hIDPSCs. These differences seemed to be related to the donor tooth conditions (deciduous or permanent, retained or erupted, and degree of root reabsorption). (J Endod 2010;36:826-831)

Eag1 potassium channel immunohistochemistry in the CNS of adult rat and selected regions of human brain

Eag1 (K(v)10.1) is the founding member of an evolutionarily conserved superfamily of voltage-gated K+ channels. In rats and humans Eag1 is preferentially expressed in adult brain but its regional distribution has only been studied at mRNA level and only in the rat at high resolution. The main aim of the present study is to describe the distribution of Eag1 protein in adult rat brain in comparison to selected regions of the human adult brain. The distribution of Eag1 protein was assessed using alkaline-phosphatase based immunohistochemistry. Eag1 immunoreactivity was widespread, although selective, throughout rat brain, especially noticeable in the perinuclear space of cells and proximal regions of the extensions, both in rat and human brain. To relate the results to the relative abundance of Eag1 transcripts in different regions of rat brain a reverse-transcription coupled to quantitative polymerase chain reaction (real time PCR) was performed. This real time PCR analysis showed high Eag1 expression in the olfactory bulb, cerebral cortex, hippocampus, hypothalamus, and cerebellum. The results indicate that Eag1 protein expression greatly overlaps with mRNA distribution in rats and humans. The physiological relevance of potassium channels in the different regions expressing Eag1 protein is discussed. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

Natural Plasmodium infections in Brazilian wild monkeys: Reservoirs for human infections?

Four hundred and forty-eight samples of total blood from wild monkeys living in areas where human autochthonous malaria cases have been reported were screened for the presence of Plasmodium using microscopy and PCR analysis. Samples came from the following distinct ecological areas of Brazil: Atlantic forest (N = 140), semideciduous Atlantic forest (N = 257) and Cerrado (a savannah-like habitat) (N = 51). Thick and thin blood smears of each specimen were examined and Plasmodium infection was screened by multiplex polymerase chain reaction (multiplex PCR). The frequency of Plasmodium infections detected by PCR in Alouatta guariba clamitans in the Sao Paulo Atlantic forest was 11.3% or 8/71 (5.6% for Plasmodium malariae and 5.6% for Plasmodium vivax) and one specimen was positive for Plasmodium falciparum (1.4%); Callithrix sp. (N = 30) and Cebus apella (N = 39) specimens were negative by PCR tests. Microscopy analysis was negative for all specimens from the Atlantic forest. The positivity rate for Alouatta caraya from semideciduous Atlantic forest was 6.8% (16/235) in the PCR tests (5.5, 0.8 and 0.4% for P. malariae, P. falciparum and P. vivax, respectively), while C apella specimens were negative. Parasitological examination of I he samples using thick smears revealed Plasmodium sp. infections in only seven specimens, which had few parasites (3.0%). Monkeys from the Cerrado (a savannah-like habitat) (42 specimens of A. caraya, 5 of Callithrix jacchus and 4 of C. apella) were negative in both tests. The parasitological prevalence of P. vivax and P. malariae in wild monkeys from Atlantic forest and semideciduous Atlantic forest and the finding of a positive result for P.falciparum in Alouatta from both types of forest support the hypothesis that monkeys belonging to this genus could be a potential reservoir. Furthermore, these findings raise the question of the relationship between simian and autochthonous human malaria in extra-Amazonian regions. (C) 2008 Elsevier B.V. All rights reserved.

Research on Candida dubliniensis in a Brazilian yeast collection obtained from cardiac transplant, tuberculosis, and HIV-positive patients, and evaluation of phenotypic tests using agar screening methods

The aim of this study was to research Candida dubliniensis among isolates present in a Brazilian yeast collection and to evaluate the main phenotypic methods for discrimination between C. albicans and C. dubliniensis from oral cavity. A total of 200 isolates, presumptively identified as C. albicans or C. dubliniensis obtained from heart transplant patients under immunosuppressive therapy, tuberculosis patients under antibiotic therapy, HIV-positive patients under antiretroviral therapy, and healthy subjects, were analyzed using the following phenotypic tests: formation and structural arrangement of chlamydospores on corn meal agar, casein agar, tobacco agar, and sunflower seed agar; growth at 45 degrees C; and germ tube formation. All strains were analyzed by polymerase chain reaction (PCR). In a preliminary screen for C. dubliniensis, 48 of the 200 isolates on corn meal agar, 30 of the 200 on casein agar, 16 of the 200 on tobacco agar, and 15 of the 200 on sunflower seed agar produced chlamydoconidia; 27 of the 200 isolates showed no or poor growth at 45 degrees C. All isolates were positive for germ tube formation. These isolates were considered suggestive of C. dubliniensis. All of them were subjected to PCR analysis using C. dubliniensis-specific primers. C. dubliniensis isolates were not found. C. dubliniensis isolates were not recovered in this study done with immunocompromised patients. Sunflower seed agar was the medium with the smallest number of isolates of C. albicans suggestive of C. dubliniensis. None of the phenotypic methods was 100% effective for discrimination between C. albicans and C. dubliniensis. (C) 2011 Elsevier Inc. All rights reserved.